【摘要】： 目的:采用不同的分離、培養(yǎng)條件,以篩選出一種相對較好的臍血單個(gè)核細(xì)胞(mononuclear cells ,MNCs)分離、培養(yǎng)方法。方法:取2007-5/2008-2新疆醫(yī)科大學(xué)第一附屬醫(yī)院產(chǎn)科健康產(chǎn)婦的臍血,比較不同分離方法所得臍血MNCs數(shù)及隨后的細(xì)胞形態(tài)學(xué)變化,比較不同換液時(shí)間、不同培養(yǎng)基、不同濃度的胎牛血清對臍血MNCs原代培養(yǎng)及傳代過程的影響,在原代培養(yǎng)第30天通過流式細(xì)胞儀檢測細(xì)胞表面免疫表型。結(jié)果:3種分離方法在分離臍血MNCs數(shù)方面差異具有統(tǒng)計(jì)學(xué)意義(P0.01),其中以羥乙基淀粉沉淀法得到的MNCs數(shù)量最多(15.23±4.30)×106/mL,密度梯度離心法、CD133免疫磁珠組所獲MNCs數(shù)分別為(3.71±1.14)×106/mL、(0.066±0.027)×106/mL。但羥乙基淀粉沉淀法所得細(xì)胞生長狀態(tài)欠佳。首次第5天～7天換液、低糖DMEM、IMDM培養(yǎng)基有利于MNCs的生長。還不能認(rèn)為30%胎牛血清與20%胎牛血清的培養(yǎng)基在培養(yǎng)臍血MNCs方面有差別。流式檢測密度梯度離心法、CD133免疫磁珠組CD34陽性率分別為10.1%、0.5%。結(jié)論:羥乙基淀粉沉淀法是一種高效的臍血分離方法,但其所得細(xì)胞的生長狀態(tài)欠佳,需進(jìn)一步實(shí)驗(yàn)證實(shí)。CD133免疫磁珠法所獲細(xì)胞純度高,可根據(jù)不同要求選用相應(yīng)分離法。首次第5天～7天換液、低糖DMEM、IMDM培養(yǎng)基是相對較好的MNCs培養(yǎng)條件。30%胎牛血清與20%胎牛血清的培養(yǎng)基在培養(yǎng)臍血MNCs方面尚未發(fā)現(xiàn)差別。
[Abstract]:Objective: to select a relatively good method for (mononuclear cells, MNCs) isolation and culture of umbilical cord blood mononuclear cells (UCB) by using different isolation and culture conditions. Methods: umbilical cord blood was collected from healthy parturients in the first affiliated Hospital of Xinjiang Medical University from May 2007 to February 2008. The number of umbilical cord blood (MNCs) obtained from different isolation methods and the subsequent morphological changes were compared, and the time of fluid exchange and different media were compared. The effects of different concentrations of fetal bovine serum on the primary culture and passage of umbilical cord blood MNCs were investigated by flow cytometry on the 30th day of primary culture. Results: there were significant differences among the three methods in the number of MNCs isolated from umbilical blood (P0.01), in which the MNCs obtained by hydroxyethyl starch precipitation method was the largest (15.23 鹵4.30) 脳 106 mL, and the density gradient centrifugation method was used. The MNCs numbers in CD133 immunomagnetic beads group were (3.71 鹵1.14) 脳 10 ~ (6) / mL and (0.066 鹵0.027) 脳 10 ~ (-6) / mL, respectively. However, the growth state of the cells obtained by hydroxyethyl starch precipitation was not good. For the first 5 ~ 7 days, the low sugar DMEM,IMDM medium was beneficial to the growth of MNCs. The culture medium of 30% fetal bovine serum and 20% fetal bovine serum could not be considered to be different in the culture of MNCs in umbilical cord blood. The positive rates of CD34 in CD133 immunomagnetic beads group were 10. 1% and 0. 5% respectively. Conclusion: hydroxyethyl starch precipitation method is an effective method for the separation of umbilical cord blood, but the growth state of the cells obtained is poor, which needs to be confirmed by further experiments. The CD133 immunomagnetic beads method has high purity and can be separated according to different requirements. For the first 5 ~ 7 days, low glucose DMEM,IMDM medium was a relatively good medium for MNCs culture, and there was no difference between 30% fetal bovine serum medium and 20% fetal bovine serum medium in the culture of umbilical cord blood MNCs.
【學(xué)位授予單位】：新疆醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2008
【分類號】：R329

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